It has been demonstrated in our initial innovation vaccine grant that replication-competent rabies virus (RV) vaccine strain-based vectors stably expressing HIV-1 gp160 can be developed and utilized. Moreover, these vectors induce a strong humoral response indicated by high titers (1:800) of HIV-NL4-3 neutralizing antibodies in mice. The recombinant RV expressing HIV induced a strong, long-lasting cytotoxic T-cell response against both HIV-1 NL4-3 and HIV-1 89.6 Env, indicating the successful priming against conserved gp160 epitopes by RV vectors. RV vectors have advantages over other viral vectors as they can: i) express stably large foreign genes, ii) have modular genome organization which make genetic manipulations easy, and iii) there is no evidence for recombination or integration. In addition, RV replicates very efficiently in most mammalian cells without killing the cells. We hypothesize that long-term expression of HIV-1 proteins without killing the cell by the viral expression vector, results in long-term presentation of HIV-1 antigen in association with both MHC class I and class II molecules. Of note, the utilized RV vector is apothogenic in a wide range of animal species and was successfully used for oral immunization against RV in chimpanzees. Our preliminary data are encouraging and the goal of this project is to continue the development of a rabies virus-based expression vector as an HIV-1 vaccine. We will expand the construction of RV vectors expressing HIV-1 and SIV Env and Gag proteins. In addition, we were able to construct recombinant RVs, where we replace the single RV envelope protein with that of vesicular stomatitis virus (VSV). These vectors will now allow boosting the immune response against HIV or SIV proteins by a second productive viral infection. A new approach for an HIV-1 vaccine is the RV glycoprotein-deleted RV expressing HIV-1 Env. These chimeric RV/HIV viruses are only infectious for cells expressing CD4 and an HIV-1 co-receptor, thus mimicking HIV-1 tropism. A virus targeted to the same target cells as HIV-1 may induce a strong immune response without the risk of an attenuated lentivirus. We will continue to analyze these recombinant RVs for their safety and potency in inducing a humoral and cellular immune response in mice. The most promising recombinant RVs expressing HIV-1 or SIV proteins will be used to immunize rhesus macaques by intra-nasal and intra-muscular routes. After detectable immunity is induced by the vaccine, the immunized monkeys will be challenged intravaginally with SIVmac251. Thus, complementary approaches will be used in vaccine development.